The present invention relates to the therapeutic and scientific uses for the human H4-1BB protein, its ligands, and the development of monoclonal antibodies that recognize and bind the H4-1BB receptor protein.
The immune system of humans and other species require that white blood cells, which include phagocytes, T lymphocytes and B cells, be made in the bone marrow. The phagocytes include macrophage cells which scavenge unwanted materials, such as virus proteins or bacterial cell walls from the system. The lymphocytes include helper T cells, killer T cells and B cells, as well as other types of cells, including those categorized as suppressor T cells. The B cells produce the antibodies. The killer T cells physically destroy target cells and the helper T cells facilitate the whole process. The complexities of the immune system and its function is facilitated, at least in part, by the lymphokines.
Lymphokines are signal transduction proteins by which the immune cells communicate with each other. Scientists have been able to produce them in sufficient quantities for therapeutic use against immunologic diseases. There are many known lymphokine proteins and they include the interferons, interleukins-1,2,3,4,5,6,7, colony-stimulating factors, lymphotoxin, tumor necrosis factor and erythropoietin, as well as others.
Interleukin 1 (IL-1), secreted from macrophages activates the helper T cells and acts to raise body temperature, causing fever, which enhances the activity of the immune cells. The activated helper T cells produce Interleukin 2 (IL-2), which in turn stimulates the helper and kdller T cells to grow and divide. The helper T cells also produce another lymphokine, B cell growth factor (BCGF), which causes B cells to multiply. As the number of B cells increases, the helper T cells produce another lymphokine known as the B cell differentiating factor (BCDF), which instructs some of the B cells to stop replicating and start producing antibodies.
T cells also produce gamma interferon (IF), which is similar to Interleukin 2 in that it has multiple effects. Gamma interferon helps activate killer T cells, enabling them to attack the invading organisms. Like BCGF, gamma interferon increases the ability of the B cells to produce antibodies. IF also keeps the macrophages at the site of the infection and helps the macrophages digest the cells they have engulfed. Gathering momentum with each kind of lymphokine signal between the macrophages and the T cells, the lymphokines amplify the immune system response such that the virus protein, an infected cell, or other foreign matter is overwhelmed and removed from the system. There are many lymphokines, maybe a hundred or more, which participate in the complex web that is the immune process. Many lymphokines and their precise effects remain unknown.
Lymphokine activities are produced when a certain lymphokine binds to its specific receptor on the surface of a target cell. Among scientists there is widespread use of cloned cell lines for production of lymphokines and their receptors. The isolation of lymphokine and lymphokine receptor messenger ribonucleic acid (mRNA) has become a common technique. The mouse receptor protein, 4-1BB, was isolated and identified based on specific expression of the T cell genes using a technique identified by the present inventor in a prior publication (Proc. Nati. Acad. Sci. USA. 84, 2896-2900, May 1987, Immunology). The protocol reported in this publication can be used by scientists to detect virtually all of the lymphokines. The method is designed to detect virtually all mRNA expressed differentially. Importantly the mRNA sequences of immune cells are expressed differentially as they relate to T cells generally, and to the killer T cells specifically. Even though the level of expression is low and the quantity of the lymphokine and its receptor protein is low, this expressed mRNA can be detected and isolated. The present inventor believes that the analysis described in the above identified publication can reveal biologically important molecules such as lymphokines and their receptors because there are many indications that biologically important or active molecules are initiated by cellular signals induced by very scarce message molecules (i.e. IF, interleukins, Map Kinase Kinase, etc.,).
Most T cell factors have been classically identified by recognizing biologic activities in assays, and thereafter purifying the protein information. An alternative approach is to isolate putative T cell genes based upon specific expression, insert them into an appropriate expression vector, and then demonstrate the function of the unknown isolated protein. Using the aforesaid modified differential screening procedure, the present inventor cloned a series of T cell subset-specific complementary deoxyribonucleic acid (cDNA) from cloned helper T lymphocyte (HTL) L2 cells, and cloned cytolytic T lymphocyes (CTL) L3.
T cells are critically important in long term acquired immunity, providing protection against viral, bacterial and parasitic infection. T cells are activated when they encounter a peptide from the invading pathogen in context with self-MHC (Major Histocompatibility Complex) via the T cell""s own T cell receptor (TCR) complex and other co-stimulatory molecule(s), such as CD-28, or CD-3. Without the engagement of the other co-stimulatory molecule(s) the T cell is rendered anergic (Vassali et al., 1979 PNAS). To date, the best-characterized co-stimulatory molecule has been CD-28. More recently, however, other cell surface molecules have been suggested to, play a co-stimulatory role, such as the molecule 4-1BB. The 4-1BB protein is a 55 kDa homodimeric molecule expressed on activated T cells in the mouse, and is a member of the Nerve Growth Factor receptor (NGFR)/Tumor Necrosis Factor receptor (TNFR) gene super family (Haskins et al., 1983, J. Exp. Med.). This family is characterized by the presence of cysteine-rich motifs in the extracellular domains. Other members of this family include NGFR, B cell activation molecule CD40, the T cell activation molecule OX-40 in rat and CD27, the two receptors for tumor mecrosis factor (TNF) called TNFR-1and TNFR-11, the apoptotic inducing protein Fas, and CD-30 which plays a role in the regulation of cellular growth and transformation.
Some of these members have been shown to play important roles in human immunodeficiency virus-1 (HIV-1) infection, including CD4+T cell proliferation, apoptosis and virus replication. The presence of high serum levels of CD30 has become a predictor of progression to acquired immunodeficiency syndrome (AIDS) although no circulating CD30 cells have been found in HIV-1 seropositive individuals. The expression of HIV-1 was induced by triggering CD30 of HIV-1 infected CD4+T cells through a nuclear factorkB (NF-kB)-dependent pathway. In HIV-1 individuals, high levels of Fas expression were observed in peripheral blood lymphocytes. Fas production was found to trigger or induce marked apoptosis of T lymphocytes, which might contribute to the CD4+T cell depletion by HIV-1 infection. The ability of CD4+T cells to express the CD40 ligand after in vitro stimulation is not impaired because of HIV-1 infection, but CD40/CD40 ligand interaction regulates HIV-1 replication of B cells in vitro. CD27 signaling enhanced proliferative response of T cells to the normal extent in HIV-1-infected individuals.
In the experiments that led to the development of this invention, a series of T cell subset-specific cDNAs were isolated from cloned murine T-cells by employing a modified differential screening procedure. The nucleotide sequence and expression properties of some of the cDNA species have been reported. One of the genes not previously characterized, which encodes mouse receptor protein 4-1BB, was studied further. These studies have led to the isolation of the human homologue to 4-1BB, H4-1BB, as well as to a series of monoclonal antibodies capable of binding the H4-1BB receptor protein and acting thereby as agonists or antagonists of H4-1BB.
T cells interact with components of the extracellular matrix (ECM) through members of the integrin family after transendothelial migration during homing to sites of inflammation. Integrin molecules are very late antigens (VLA""s) in a family of cell surface receptors that mediate the adhesion of cells to ECM proteins as well as other cells. The heterodimeric integrins comprising of various alpha and beta subunits, act as a transducing mechanism of extracellular signals. Regulation of integrin function is utilized by T cells and other leukocytes for rapid adhesion following activation of the cells.
The major factors known so far to affect the differentiation of the T cells are the lymphokines (also referred to as cytokines), such as IL-2 and IL4. In vitro and in vivo studies with transgenic mice have demonstrated that IL-2 induces the development of the Th1 subset of T cells by priming them for efficient IF production and preventing development of IL-4-producing cells. Previously however, it was unknown how their interactions worked to direct the amplification of the immune response and development of the Th1 or Th2 subset of T cells.
Specific immune responses are governed by the recognition of antibodies to foreign antigens. Antibodies form a family of structurally related glycoproteins and confer, generally to the organism producing them, the protective effect of cell-mediated immunity. Antibodies are produced by B-lymphocytes and are bound to the cell membrane, functioning as B cell receptors for antigens. Antibodies are also secreted by B cell progeny that differentiate in response to stimulation by antigens. A specific antigen will trigger the complementary B lymphocyte(s) to proliferate and differentiate into effector cells, which then eliminate the antigen. Each lymphocyte produces an antibody of a particular specificity, and thus immune responses are very specific for distinct antigens. The portion of the antigen recognized by T and B lymphocytes are called epitopes or determinants.
The development of techniques to produce virtually unlimited amounts of a single (monoclonal) antibody for a specific antigenic epitope has had an enormous impact on clinical immunology. To produce a monoclonal antibody (mAb) of known specificity, a mouse can be injected with a particular antigen, such as a receptor protein and the spleen B lymphocytes (that produce the antibody against the protein) can be fused via somatic cell hybridization to a myeloma (lymphocyte tumor) to produce an immortal cell line to create a hybridoma. This is done because normal B-lymphocytes can not grow indefinitely, yet when fused with the myeloma, the resulting hybridoma produces a virtually endless supply of a specific monoclonal antibody. Selection techniques have been developed to ensure that only the fused cells continue to grow. Each hybridoma cell is specific for only one antigenic determinant. If several different antibody producing hybridomas are produced, each hybridoma clone of an individual B lymphocyte will secrete an antibody for only one surface antigenic determinant. To determine which mAbs specifically bind to the protein receptor, or which has a desired activity (e.g. the mAb acts as an agonist, antagonist, or has the most specific binding to a critical epitope), the hybridomas can be screened with an ELISA (enzyme-linked immunosorbent assay).
Monoclonal antibodies have numerous applications: 1) The hybridoma can produce large quantities of specific antibodies that are normally either unavailable in small quantities or not available at all; 2) the hybridoma can be directed to produce antibodies against a single antigen determinant which, for complex antigens, may be normally very difficult; 3) pure antibodies can be obtained against antigens that cannot be purified; 4) immuno-diagnosis of infectious and systemic diseases by detecting specific antigens circulating in tissues or using monoclonal antibodies in immunoassays; 5) characterization of protein receptors and the role they play in the transition from a naive to a memory T cell; and 6) blocking or enhancing immune response or activation.
The invention below presents uses for the H4-1BB protein, its ligands, antibodies thereto and other co-stimulatory molecules that can be used therapeutically in the treatment of cancer and HIV-1.
The present invention includes the human receptor protein H4-1BB and the cDNA gene encoding for human receptor protein H4-1BB. The nucleotide sequence of the isolated cDNA is disclosed herein along with the deduced amino acid sequence. The cDNA gene identified as pH4-1BB was deposited at the Agricultural Research Service Culture Collection and assigned the accession number: NRRL B21131
The cDNA, including its fragments and derivatives, can be used as a probe to isolate DNA sequences encoding for proteins similar to the receptor protein. The cDNA of the human receptor, H4-1BB, was isolated by using probes derived from cDNA 4-1BB. The cDNA gene identified as p4-1BB was deposited at the American Type Culture Collection at 10801 University Bvld., Manassas, Va. 20110-2209 under ATCC No.: 67825 The present invention also provides an antibody specific for H4-1BB. On exemplary hybridoma which secretes such an antibody was deposited at the American Type Culture Collection under ATCC No. HB-11860.
The human receptor protein H4-1BB can be produced by: 1) inserting the cDNA of H4-1BB into an appropriate expression vector, 2) transfecting the expression vector into an appropriate transfection host, c) growing the transfected hosts in appropriate culture media and d) purifying the receptor protein from the culture media. The protein and fragments and derivatives can be used: 1) as a probe to isolate ligands to human receptor protein H4-1BB, 2) to stimulate proliferation of B-cells expressing H4-1BB ligands, or 3) to block H4-1BB ligand binding.
Bell proliferation can be induced by treating B-cells that have expressed a ligand to receptor protein H4-1BB with cells that have expressed receptor protein H4-1BB. The use of H4-1BB protein, H4-1BB ligand protein, or fragments of the proteins, to block H4-1BB ligand binding has practical application in the suppression of the immune system during organ transplantation.
Monoclonal antibodies generated against H4-1BB can be used to enhance or suppress T-cell proliferation and activation by treating T-cells that have expressed receptor protein H4-1BB with an anti-H4-1BB monoclonal antibodies. To enhance immune reaction antibodies which act as agonists can be generated, to suppress T-cell proliferation and/or activation antibodies which act as antagonists can be generated. To this end, four monoclonal antibodies have been developed for use. The monoclonal antibodies BBK-1 and BBK-4 are agonists to receptor protein H4-1BB, while monoclonal antibodies BBK-2 and BBK-3 are antagonists to receptor protein H4-1 BB, and can be used to either upregulate the immune system or suppress its activity. Some tumors are potentially immunogenic but do not stimulate an effective anti-immune response in vivo. Tumors may be capable of delivering antigen-specific signals to T cells, but may not deliver the co-stimulatory signals necessary for fill activation of T cells. A monoclonal antibody generated against H4-1BB (e.g. an agonist) is capable of eradicating tumors with low immunogenicity by providing for the full activation, enhancement, and/or proliferation of T-cells. Moreover, an anti-H4-1BB mAb agonist has great utility in assessing the role of the 4-1BB receptor protein in the transition from naive to memory T-cells. Cross-linking of the 4-1BB with an anti-H4-1BB mAb agonist, such as BBK-1 or BBK-4 will produce the effects similar to the binding of the 4-1BB ligand to 4-1BB.
An antagonistic H4-1BB mAb can also be used to interfere with H4-1BB and H4-1BB ligand binding. By interfering with ligand binding, as with the use of an anti-H4-1BB mAb antagonist BBK-2, and BBK-3, the immune responses will be suppressed. In this context, diseases that would benefit from the therapeutic use of such a mAb include rheumatoid arthritis, systemic lupus erythematosus, and diabetes. Alternatively this type of molecule is useful in organ transplantation to suppress immune system mediated rejection of transplanted tissue.
A fusion protein can detect cell membrane ligands to human receptor protein, H4-1BB. A fusion protein of the present invention comprises the extracellular portion of the receptor protein H4-1BB and a detection protein (alkaline phosphatase) or Fc portion of an IgG1 bound to the portion of the receptor protein H4-1BB. In addition, this disclosure demonstrates that co-engagement of CD28 with 4-1BB promoted type 1 effector T cell development. The 4-1BB signal regulated CD28 mediated cytokine production profiles in two ways, enhancing type 1 and, at the same time, suppressing type 2 cytokine (lymphokine) production. The 4-1BB-mediated co-stimulation also induced xcex3-interferon (IF) production in Th1 cells. The expression of 4-1BB was subset-specific, being detected predominantly on IF producing, but not on IL-4-producing cells. Moreover, it was determined that 4-1BB and CD30 expression were mutually exclusive, representing type 1 and type 2 subsets, respectively. The co-engagement of 4-1BB with CD28 enhanced long-term cell survival for cells susceptible to apoptosis induced by repeated TCR activation. Therefore, it was demonstrated that 4-1BB and CD30 interplay to regulate the balance between type 1 and type 2 T cell subsets, and the polarization of the immune response. Therapeutically they can be used to achieve opposite effects.
This invention presents data, which demonstrates that there is a functional correlation between 4-1BB and CD28 in T cell adhesive responses. The inventors disclose herein that 4-1BB can induce of cell adhesion. Enhanced cell adhesion through the presence 4-1BB signal or agonist results in a maximal T cell activation in response to repeated exposure to sub-optimal concentrations of anti-CD3 and anti-CD28. Thus, 4-1BB effects were attributed by its ability in reducing the threshold of anti-CD3 concentration needed; to repeatedly activate primary T cells. Therefore the degree of cell adhesion in response to anti-4-1BB correlated with the 4-1BB expression levels, and correspondingly effects the therapeutic use of these molecules.
The level of 4-1BB expression and the percentage of 4-1BB-expressing T cells was higher in HIV-1 positive individuals than in the HIV-1 controls (P less than 0.01). 4-1BB signal cooperated with CD28 to induce HIV-1, and CD4+T cell proliferation. In addition, cross-linking 4-1BB with agonistic monoclonal antibodies enhanced HIV-1 replication both in primary stimulation and secondary re-stimulation of CD4+T cells from HIV-1 individuals. Thus, 4-1BB is involved in the activation of HIV-1 replication from latently infected CD4+T cells, and the 4-1BB co-stimulatory pathway can be the target of therapeutic intervention. If the pathway of HIV-1 infection is disturbed at the early stages of the infection, through the use of H4-1BB antibodies a lower virus load will result. Moreover, the increased 4-1BB expression on CD8+T cells is noteworthy because it is correlated with the degree of immunodeficiency in HIV-1 infection, cross-linking 4-1BB on CD8+T cells will induce enhanced cytotoxic activity against HIV-1 infected CD4+T cells.
An object of the present invention is to teach a fission protein comprising the extracellular portion of H4-1BB and a detection protein.
Another object of the present invention is to teach the method of constructing a monoclonal antibody against H4-1BB.